Hepatitis C virus (HCV) is a major cause of chronic liver diseases, hepatic cirrhosis and hepatocellular carcinoma. More than half of HCV-infected patients develop chronic hepatitis which leads to lethal cirrhosis or hepatoma. It is reported that approximately 170 million people, more than 3% of total earth population, are infected with HCV (Miller and Purcell, Proc. Natl. Acad. Sci. USA, 87: 2057-2061 (2000)). The conventional treatment method for HCV-infected patients involves the administration of interferon (α-interferon) or ribavirin. However, just about 50% of these patients respond to interferon, and 50% of the treated patients suffer from relapsed hepatitis C (Hino et al., J. Med. Virol. 42(3):299-305 (1994); Tsubota et al., Hepatology. 19(5):1088-94 (1994)). Another problem of interferon-based treatment is the high cost of the treatment which requires hospitalization. Although ribavirin, one of nucleic acid derivatives, works effectively for 40 to 70% of acute hepatitis patients, it is not effective for chronically HCV-infected patients.
Any successful treatment or preventive method for HCV infection or HCV-mediated chronic liver disease has not yet been developed. Therefore, there remains a need to develop a HCV-specific antiviral agent.
HCV belongs to Flaviviridae family causing non-A non-B hepatitis. HCV genome comprises a single-stranded RNA which expresses a polyprotein composed of 3,010 amino acids (Choo et al., Science, 244:359-362 (1989)). The HCV polyprotein is cut by virus or host cell proteases into 10 functionally different proteins. HCV is composed of a row of genes, namely NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (Steven Rosenberg, J. Mol. Biol., 313:451-464 (2001)), which are classified into two groups: one, structural proteins including C (core), E1, E2 and p7 and the other, non-structural proteins including NS2, NS3, NS4A, NS4B, NS5A and NS5B.
C (core) protein of HCV is believed to be responsible for the encapsidation of HCV genomic RNA and to play an important role in the development of hepatoma by regulating gene transcription, growth and proliferation of host cells. E1 and E2 proteins are type-1 transmembrane and virus envelope proteins, known to be involved in cell infection. E1 protein had not been studied in detail in the past because of its incapability of inducing neutralizing antibody. Recently, however, a therapeutic vaccine using E1 has been developed by Innogenetics, Co. (Belgium), which is in the process of phase II clinical trial following successful phase I clinical trial with chimpanzees. It is encouraging that E1 protein can be effectively used for the treatment of 1b type HCV infection which has not been successfully treated by alpha-interferon. E2 protein, one of critical envelope proteins of HCV, has been known as a multi-functional protein which conjugates with the assumed cell receptor CD81, and it nullifies both the immune system's ability to action of a host cell and interferon-mediated antiviral reaction, which leads to oncogenesis or autoimmune liver disease. Thus, E2 is a major target for the development of an anti-HCV agent. The function of P7 protein has not yet been disclosed. NS2 protein is a part of a metallo-protease, and NS3 harbors serine protease of HCV at its N-terminus and RNA helicase domain of its C-terminus. NS4A is a cofactor of viral protease, and NS4B has been found by the present inventors to have a potential for tumorigenesis. NS5A functions to endow HCV resistance against interferon and antiapoptosis. NS5B acts as a viral RNA-dependent RNA polymerase. Non-structural proteins including NS2, NS3, NS4B and NS5B have also been the major targets for the development of antiviral agent inhibiting viral replication.
Cytotoxic T lymphocytes (CTLs) play an important role in eradicating virus from a virus-infected individual. Once infected with HBV, a patient develops an acute hepatitis. However, in most acute hepatitis B patients, a powerful polyclonal CTL response occurs, leading to the natural cure of the disease, but a significant CTL response cannot be induced in chronic hepatitis B patients.
Even when HCV-specific CTL response is induced in the liver and peripheral blood of chronically HCV-infected patients, such CTL response is too weak to get rid of the virus effectively. Nevertheless, a number of research efforts have showed that HCV infection can be controlled by HCV-specific CTL response, suggesting that it is possible to develop an effective antiviral therapy based on the amplification of HCV-specific CTL response.
An epitope inducing antiviral CTL response can induce very effective cellular immunity for the prevention or treatment of viral infection. The use of an antigen-specific epitope or a DNA construct encoding the epitope is more advantageous than that of a conventional whole protein antigen. First, it is safe. Second, there is no likelihood of reducing an immune response due to mutation, unlike the case when a whole protein antigen is used. Third, it can be produced easily. Fourth, it is easy to produce a multi- or poly-vaccine using epitopes originating from multiple antigens of pathogens.
However, epitope-based vaccine and the treatment therewith has been limitedly applied due to the polymorphism of HLA (human leukocyte antigen) (hereinafter, also referred to as “MHC (major histocompatibility complex)”) and the diversity of the epitopes binding to HLA subtypes. Up to now, most studies on antigen-specific antiviral CD8+ T cell response have been focused on HLA-A2 positive patients.
To induce a cellular immunity, an epitope peptide must be exposed on the surface of an antigen-presenting cell (APC) for forming a complex with MHC on APC. When T cell receptors (TCRs) on the surfaces of T cells recognize the structure of the MHC-peptide complex, the information on the recognized antigen is transferred to the T cells, which are activated by the interaction of CD40 with CD40L, the ligand of CD40 on the surfaces of APCs. Signal transduction through the CD40-CD40L interaction stimulates APCs, leading to the expression of co-stimulatory molecules such as B7-1 and B7-2. As a result, T cells can function as effector T cells having active molecules like CD28, 4-1BB, and CD25.
In chronic hepatitis patients, the expression of co-stimulatory molecules, which are known to promote a CTL-mediated immune response, is reduced and the maturation of dendritic cells known as APCs is inhibited, while the function and the number of natural killer cells become low. In this regard, it is expected that the administration of such a co-stimulatory molecule can enhance cellular immunity. Thus, the present inventors demonstrated that the cellular immunity can be enhanced by such a cofactor as: CD40LT, a trimer form of CD40L which induces the maturity of dendritic cells; 4-1BBL which increases the density of CD8+ T cells and promotes the functions of memory T cells; IL-15 and FLT-3L which induce the maturity of dendritic cells and promote the functions of natural killer cells that are depleted in HCV-infected patients; B7-1 and B7-2 which play important roles in recognizing epitope antigen; and HSP (Heat shock protein) which improves the epitope-presenting process.
The use of a peptide antigen is safer and more effective than the use of a whole protein antigen, but has some limitations in that the costs for peptide synthesis and purification are high and a specific antigen delivery system is required. An exogenous peptide antigen cannot be expected to match the entire processes of epitope generation from endogenous antigen like tumor antigen or viral antigen. The antigenicity is significantly affected by the physical properties of the peptide, while a DNA antigen has advantages over a peptide antigen such as: the production cost is low; the easiness of handling; and its effectiveness in process of epitope generation inside cells without any special means for the presentation and recognition of tumor or viral antigens. It is reported that a DNA antigen induces an effective cell-mediated immune when it is provided in the form of a eukaryotic expression vector (Cara C Wilson et al. J. Immunol., 171:5611-5623 (2003)).
To overcome the above-mentioned limitations of the conventional epitope-based immunotherapy and to induce a strong HCV-specific CTL response, the present inventors have designed multi-HLA supertype reactive epitopes from the conservative regions of HCV polyprotein through motif search, and found that the epitopes induce an effective HCV-specific immune response by their binding to not only HLA-A2 type but also other HLA-A and HLA-B types. The present inventors have also found that when an expression vector including an epitope-encoding oligonucleotide or polynucleotide is administered to an animal model, an effective HCV-specific immune response can be induced.